Circulating Levels of LAMP2 in Coronary Artery Disease: Association with Serum Lipid Profile.

Whereas several in vitro and in vivo studies have described the role of lysosomal associated membrane protein 2 (LAMP2) in lipid homeostasis, there is no study addressing LAMP2 serum concentration and its association with lipid profiles in the context of coronary artery disease (CAD). We aimed to determine the LAMP2 serum concentration and its association with serum lipid profiles as well as the gene expression of LAMP2 in peripheral blood mononuclear cells (PBMCs) of CAD patients and control group. Circulating levels of LAMP2 were quantified by enzyme-linked immunosorbent assay (ELISA) in CAD patients (n=85) and control group (n=65) and correlation to lipid parameters was assessed. Gene expression analysis was performed by quantitative real-time PCR. Mean LAMP2 serum concentration adjusted for drug consumption, age and gender was not significantly different between the CAD and control groups (p>0.05). However, LAMP2 serum concentration showed independent significant association with lipid profiles including triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) (all p<0.05). Furthermore, increased expression of LAMP2 has been observed in PBMCs of CAD patients compared to the control group (p<0.05). Our findings supported the previous observations showing the contribution of LAMP2 in lipid homeostasis and pathogenesis of CAD.

Comparing acute and chronic human cutaneous leishmaniasis caused by Leishmania major and Leishmania tropica focusing on arginase activity.

BACKGROUND: Cutaneous leishmaniasis (CL) in Iran is mainly caused by Leishmania major (L. major) and L. tropica. Arginase mediated L-arginine metabolism is an important issue in Leishmania parasite propagation. Arginase activity in human CL due to L. major and L. tropica have not been studied up to now. OBJECTIVES: We aimed to compare the clinical and laboratory aspects of acute and chronic CL, focussing on arginase activity. METHODS: In this case-control study, 30 patients with acute CL (duration ≤ 1 year), 13 patients with chronic CL (duration ≥ 2 year) and 11 healthy controls were recruited. Arginase activity was measured in skin biopsies of lesions, peripheral blood polymorphonuclear cells (PMNs), peripheral blood mononuclear cells (PBMCs) and plasma by standard methods. RESULTS: The median of arginase activity in the acute lesions was higher than in chronic samples and significantly higher than in healthy controls (P = 0.008). PMNs of both acute and chronic patients showed higher levels of arginase activity as compared to the levels in PBMCs and plasma. The median of arginase activity in the PMNs of patients with chronic CL was higher than that of patients with acute CL and significantly higher than that of the healthy controls (P = 0.010). CONCLUSION: The level of arginase activity in lesions of patients with acute and chronic CL was higher than the skin of healthy controls. The highest level of arginase activity was observed in PMNs from patients with chronic CL. This suggests that the high level of arginase activity in PMNs of patients with chronic CL may contribute to the chronicity.

Comparison of the Antimicrobial Properties of Silver Impregnated Vascular Grafts with and without Triclosan.

OBJECTIVES: The aim was to compare the antimicrobial efficacy of the silver impregnated collagen coated polyester vascular graft (IGS) with an identical graft combining silver and triclosan (IGSy). METHODS: This was an in vitro study. A non-antimicrobial collagen polyester vascular graft served as control (IG). The IG, IGS, and IGSy grafts were contaminated separately with inoculates of each of the following micro-organisms: Staphylococcus epidermidis (SE), methicillin resistant Staphylococcus aureus (MRSA), and Escherichia coli producing extended spectrum beta-lactamase (ESBL-EC) or Candida albicans (CA). MRSA, ESBL-EC, and CA were obtained from retrieved infected grafts. The in vitro antimicrobial efficacies of the contaminated grafts were evaluated by time to kill assays over a 24 hour period in accordance with CLSI Guideline M26-A. All assays were repeated six times. Bacterial survival numbers were obtained at 1, 4, 8, and 24 hours using a standard plate count procedure. Bactericidal activity was defined as a 3 log10 reduction factor (logRF). To calculate the overall difference in the mean log10 CFU/mL within 24 hours, a one way ANOVA with a Bonferroni correction was calculated separately for each graft. RESULTS: The IG graft showed an increase in the number of viable organisms for the four strains tested. IGSy offered better antimicrobial properties than IGS for both ESBL-EC and MRSA, since only the IGSy graft achieved > 3 logRF and fulfilled the standard criteria for bactericidal activity at 24 hours with 3.78 and 4.08 logRF, respectively. For samples inoculated with SE and CA, both antimicrobial grafts achieved 24 hour bactericidal activity with > 3 logRF. However, for CA the one-way ANOVA analysis demonstrated that the IGSy graft performed differently in terms of speed of antimicrobial action, appearing more active as early as 4 hours following inoculation (p = .007). CONCLUSION: In the in vitro conditions, the Synergy vascular graft combining silver with triclosan demonstrated better short-term antimicrobial activity than the silver graft for all micro-organisms tested.

Description of an original integron encompassing blaVIM-2, qnrVC1 and genes encoding bacterial group II intron proteins in Pseudomonas aeruginosa.

OBJECTIVES: A burn unit of a hospital in Tunis underwent an endemic situation caused by imipenem-resistant Pseudomonas aeruginosa. For nine non-repetitive isolates of a clonal VIM-2-producing strain, the blaVIM-2 genetic background was characterized and the associated qnrVC1 gene molecularly analysed. METHODS: The imipenem resistance mechanism was investigated by phenotypic and molecular tests, and resistance transfer was studied by conjugation and transformation experiments. The integron's structure was characterized by sequencing, and qnrVC1 expression was explored after cloning experiments. RESULTS: The nine VIM-2-producing strains were collected from eight patients and one environmental sample. All transfer assays failed, suggesting a chromosomal location of blaVIM-2. This latter was found to be part of a class 1 integron of ∼7500 bp, which also contains blaOXA-2, aadA1 and two copies of the aadB, arr-6 and qnrVC1 genes. qnrVC1 exhibited higher homology with the chromosomally encoded qnr genes of Vibrionaceae than with plasmid-mediated qnr genes of Enterobacteriaceae. The qnrVC1 gene cassette possesses a promoter allowing its expression, and it conferred decreased fluoroquinolone susceptibility to Escherichia coli. Additionally, on the same integron, genes encoding an uncommon group IIC-attC intron were detected. CONCLUSIONS: A VIM-2-producing P. aeruginosa outbreak led us to characterize an integron harbouring a qnrVC1 cassette and a new group IIC-attC intron. This is the first known description of a qnr determinant in a P. aeruginosa strain. Its presence conferred a low level of resistance to quinolones in E. coli, which might favour the emergence of highly resistant mutants.

The Effect of Sub-MIC ß-Lactam Antibiotic Exposure of Pseudomonas aeruginosa Strains from People with Cystic Fibrosis in a Desiccation Survival Model.

Prior to modern typing methods, cross-infection of P. aeruginosa between people with cystic fibrosis (CF) was felt to be rare. Recently a number of studies have demonstrated the presence of clonal strains of P. aeruginosa infecting people with CF. The aim of this study was to determine whether strains of P. aeruginosa demonstrated differences in resistance to desiccation and whether preincubation in subminimum inhibitory concentrations (MICs) of ß-lactam affected desiccation resistance. The experimental data were modelled to a first-order decay model and a Weibull decay model using least squares nonlinear regression. The Weibull model was the preferred model for the desiccation survival. The presence of a mucoid phenotype promoted desiccation survival. Preincubation with antibiotics did not have a consistent effect on the strains of P. aeruginosa. Meropenem reduced desiccation resistance, whereas ceftazidime had much less effect on the strains studied.

SHV-12, SHV-5, SHV-2a and VEB-1 extended-spectrum beta-lactamases in Gram-negative bacteria isolated in a university hospital in Thailand.

Sixty-one extended-spectrum beta-lactamase (ESBL)-producing isolates were collected from Srinagarind Hospital, Thailand. These included 43 Enterobacteriaceae and 18 Pseudomonadaceae. The 43 Enterobacteriaceae were found to produce the following ESBLs: 26 (60.5%) SHV-12, 13 (30.2%) SHV-5, two (4.7%) SHV-2a, one (2.3%) VEB-1 and one (2.3%) unidentified. Twenty-four isolates (55.8%) also carried bla(TEM-1B), as well as bla(SHV) or bla(VEB-1). Plasmid DNA from transconjugants carrying the bla(SHV-12) gene showed various restriction patterns, indicating the distribution of the bla(SHV-12) gene among different antibiotic resistance plasmids. In contrast, bla(SHV-5) in 13 isolates was found on a single plasmid of c. 130 kb. Pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA from these isolates revealed that nine of 11 Klebsiella pneumoniae gave the same pattern, indicating clonal spread of the strain within the hospital, together with the occasional spread of the plasmid to other strains. Among the pseudomonad isolates, 16 Pseudomonas aeruginosa and one Pseudomonas putida had bla(VEB-like) and one P. aeruginosa had bla(SHV-12). Nine of the 16 isolates carrying bla(VEB-like) (56.3%) had identical PFGE patterns, suggesting the dissemination of this gene, also by clonal spread. At least six different bla(VEB-like-)containing integrons were found among the 18 isolates. This is the first report of bacteria producing SHV-12 and SHV-2a in Thailand and the first report of SHV-12 in P. aeruginosa, of VEB-1 in Citrobacter freundii and a VEB-1-like beta-lactamase in P. putida. These findings indicate that ESBL genes in the Far East are part of a gene pool capable of broad horizontal gene transfer, in that these genes can transfer between different families of Gram-negative bacilli.

Discrimination of SHV beta-lactamase genes by restriction site insertion-PCR.

Restriction site insertion-PCR (RSI-PCR) is a simple, rapid technique for detection of point mutations. This technique exploits primers with one to three base mismatches near the 3' end to modulate a restriction site. We have developed this technique to identify described mutations of the bla(SHV) genes for differentiation of SHV variants that cannot be distinguished easily by other techniques. To validate this method, eight standard strains were used, each producing a different SHV beta-lactamase: SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, SHV-6, SHV-8, and SHV-18. Mismatch primers were designed to detect mutations affecting amino acids at positions 8 (SspI), 179 (HinfI), 205 (PstI), 238 (Gly-->Ala) (BsrI), and 240 (NruI) of bla(SHV) genes. All amplimers of the bla(SHV) genes used in this study yielded the predicted restriction endonuclease digestion products. In addition, this study also makes theoretical identification of bla(SHV-6), bla(SHV-8), and 12 novel bla(SHV) variants using the PCR-restriction fragment length polymorphism (RFLP) technique possible. By using a combination of PCR-RFLP and RSI-PCR techniques, up to 27 SHV variants can now be distinguished rapidly and reliably. These simple techniques are readily applied to epidemiological studies of the SHV beta-lactamases and may be extended to the characterisation of other resistance determinants.

Occurrence of a new metallo-beta-lactamase IMP-4 carried on a conjugative plasmid in Citrobacter youngae from the People's Republic of China.

During the course of an antimicrobial resistance surveillance programme in Guangzhou, the People's Republic of China, single strains of Citrobacter youngae and Pseudomonas aeruginosa were identified which were resistant to imipenem and found to carry the carbapenemase gene bla(IMP). PCR screening of the citrobacter strain with specific primers for the bla(IMP) type genes gave a 587-bp product which when sequenced gave 100% homology with the bla(IMP-4) sequence reported recently from Acinetobacter spp. The determinant in the C. youngae strain was found to be located on a 156-kb plasmid capable of transfer to Escherichia coli UB1637 by conjugation. Sequencing of the bla(IMP-4) open reading frame in the C. youngae strain and adjacent sequences not only confirmed the presence of bla(IMP-4) but also identified that a conserved core site found within the 59-bp element of integrons was present and the same as the one described in the only other occurrence of bla(IMP-4) in Acinetobacter spp. isolated from an intensive care unit in Hong Kong. This is the second report of transferable carbapenemase genes in Enterobacteriaciae outside of Japan and the first in the People's Republic of China. Under the selective pressure of carbapenems and extended spectrum cephalosporins use we might expect this gene to spread and widespread surveillance should be instituted.

Detection of extended-spectrum beta-lactamases in members of the family enterobacteriaceae: comparison of the MAST DD test, the double disc and the Etest ESBL.

A technically simple method-the MAST double disc (MDD) test-for the detection of extended-spectrum beta-lactamase (ESBL) production by bacteria is described. A wide range of ESBL, non-ESBL and Class 1 beta-lactamase-producing isolates was examined. The MDD test, which uses discs containing ceftazidime and a complementary disc containing ceftazidime and clavulanate and a second pair containing cefotaxime and cefotaxime and clavulanate was compared with the standard double disc diffusion test and an Etest method. Both the Etest and the MDD correctly identified 93% of ESBL producers. The MDD is an inexpensive alternative to current methods for the detection of ESBL production.

Characterisation of extended-spectrum beta-lactamases of the SHV family using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP).

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been developed to extend the identification of SHV beta-lactamases previously characterised by PCR-single strand conformational polymorphism (PCR-SSCP) analysis alone. Eight bacteria, each producing a different SHV beta-lactamase, were used in this study. These bacteria harbour bla(SHV-1), bla(SHV-2a), bla(SHV-3), bla(SHV-4), bla(SHV-5) (two strains), bla(SHV-11) and bla(SHV-12). All isolates were characterised by PCR-SSCP and PCR-RFLP with DdeI and NheI digestion. By a combination of these techniques, the genes encoding these beta-lactamases could be differentiated from each other. In addition, the PCR-RFLP technique theoretically can be applied to distinguish the genes encoding SHV-7, SHV-9, SHV-10, SHV-15, SHV-17 and SHV-24 from those encoding other SHV variants. We report a simple PCR-RFLP technique that can be used in epidemiological studies to enable the rapid characterisation of known SHV beta-lactamases in a combination with the previously published PCR-SSCP analysis.

Evolution and spread of SHV extended-spectrum beta-lactamases in gram-negative bacteria.

Resistance to beta-lactam antibiotics has been a problem for as long as these drugs have been used in clinical practice. In clinically significant bacteria the most important mechanism of resistance is the production of one or more beta-lactamases, enzymes that hydrolyse the beta-lactam bond characteristic of this family of antibiotics. Prominent among the beta-lactamases produced by the Enterobacteriaceae is the SHV family. The first reported SHV beta-lactamase had a narrow spectrum of activity. By the accumulation of point mutations at sites that affect the active site of the enzyme, a family of derivatives of SHV-1 has evolved. Derivatives of SHV-1 either have an extended spectrum of activity, capable of inactivating third-generation cephalosporins, or are resistant to beta-lactamase inhibitors. This review describes the evolution and spread of the SHV family of beta-lactamases, introducing the structure-function analysis made possible by DNA sequence analysis. It also reviews the methods used to characterize members of this family of beta-lactamases, indicating some of the difficulties involved.

PCR single strand conformational polymorphism can be used to detect the gene encoding SHV-7 extended-spectrum beta-lactamase and to identify different SHV genes within the same strain.

The PCR single strand conformational polymorphism (PCR-SSCP) technique described to identify mutants of the SHV beta-lactamases was extended to identify an SHV-7 type beta-lactamase. This was found in a strain of Klebsiella pneumoniae, the first recorded isolate in the UK to produce this type of enzyme. We also demonstrate that PCR-SSCP can be used to identify more than one SHV beta-lactamase gene in a single strain.

Transcontinental importation into the UK of Escherichia coli expressing a plasmid-mediated AmpC-type beta-lactamase exposed during an outbreak of SHV-5 extended-spectrum beta-lactamase in a Leeds hospital.

Sixteen strains of Escherichia coli with high-level resistance to extended-spectrum cephalosporins and other classes of antibiotic have been isolated at St James' University Hospital, Leeds. They produce up to three separate beta-lactamases: TEM-1, SHV-5 and, in five isolates, a plasmid-mediated AmpC-type enzyme. With the exception of carbapenems, the isolates reported in this study were resistant to all beta-lactam antibiotics including extended-spectrum cephalosporins and the monobactam aztreonam. There was evidence of the spread of a plasmid encoding SHV-5, particularly amongst patients on the liver transplant unit. Sensitivity to beta-lactam antibiotics in five isolates expressing the AmpC-type beta-lactamase was not restored by the beta-lactamase inhibitor clavulanic acid. These bacteria also carried blaSHV-5 on a large plasmid. PCR-amplification of the structural gene and digestion with restriction endonucleases demonstrated that the plasmid-mediated blaAmpC probably identified as BIL-1 using the criteria available. Four of the five patients carrying isolates that carried the plasmid-located blaAmpC gene had recently visited the Indian subcontinent and we presume that they returned carrying these bacteria. Restriction fragment length polymorphism analysis using pulsed field gel electrophoresis (PFGE) suggests that at least four distinct strains existed amongst these five isolates. The two isolates that had very similar PFGE patterns had different plasmid profiles and were isolated from different locations in the hospital and at different times. This study demonstrates the ease with which highly resistant bacteria can be imported into the UK and spread within hospitals.

Detection of mutations conferring extended-spectrum activity on SHV beta-lactamases using polymerase chain reaction single strand conformational polymorphism (PCR-SSCP).

Single strand conformational polymorphism (SSCP) is a recently developed technique used to detect single base mutations in short PCR-generated amplimers. The method has been adapted and applied to differentiation of beta-lactamase genes. Each of the five standard SHV strains used produced a unique SSCP pattern, allowing the possibility of rapid identification of the SHV genes of other isolates. A clinical isolate that phenotypically produced SHV-5 yielded a pattern of major bands indistinguishable from that of the SHV-5 standard strain, illustrating the applicability of this technique. We therefore report a reliable and reproducible technique that can be applied to the characterisation of the SHV beta-lactamases.